Presteady-State Currents of the Rabbit Na + /Glucose Cotransporter (SGLT1)

Hazama, Akihiro; Loo, Donald D. F.; Wright, Ernest M.

doi:10.1007/s002329900169

Cited by 96 publications

(182 citation statements)

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“…Maximal charge (Q max ) was conserved upon progressive removal of Na ϩ , whereas the voltage midpoint (V 0.5 ) shifted to more hyperpolarized potentials. Pre-steady-state currents have been observed for several other Na ϩ -coupled (1,9,14,17,19,27) or H ϩ -coupled (8,12,16,21,22,25) solute transporters. The results of model simulation have led investigators to attribute pre-steady-state currents to two steps in the transport cycle, namely reorientation of the empty charged carrier and binding/dissociation of the driving ion partway within the plane of the membrane electric field (and in some cases also to the binding/dissociation of the ion at the internal face).…”

Section: Discussionmentioning

confidence: 99%

“…These were isolated from capacitive transient currents (which decayed with half-times of 0.3-0.9 ms) and steady-state currents by the fitted method (9,15,21,22). The compensated currents thus obtained were integrated with time to obtain charge movement (Q).…”

Section: Heterologous Expression Of Human Svct1 In Xenopus Oocytesmentioning

confidence: 99%

“…We did not consider the inward-facing Na ϩ -bound configuration C7 ϭ [CNa]Љ since intracellular Na ϩ concentration ([Na ϩ ]) is generally low. Our partial-reaction model is essentially identical to that used to describe the Na ϩ -glucose cotransporters SGLT1 (9,14,28) and SGLT3 (formerly pSGLT2) (17) and similar to that used to describe a myo-[H ϩ ]inositol cotransporter (12). The rate of change in concentration of each carrier state is given by the difference of forward and reverse reactions…”

Section: Heterologous Expression Of Human Svct1 In Xenopus Oocytesmentioning

confidence: 99%

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Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Mackenzie¹,

Illing²,

Hediger³

2008

American Journal of Physiology-Cell Physiology

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Mackenzie B, Illing AC, Hediger MA. Transport model of the human Na ϩ -coupled L-ascorbic acid (vitamin C) transporter SVCT1. Am J Physiol Cell Physiol 294: C451-C459, 2008. First published December 19, 2007 doi:10.1152/ajpcell.00439.2007.-Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-Ascorbic acid transport was saturable (K0.5 Ϸ 70 M), temperature dependent (Q10 Ϸ 5), and energized by the Na ϩ electrochemical potential gradient. We obtained a Na ϩ -L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-Ascorbic acid and Na ϩ saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na ϩ , L-ascorbic acid, Na ϩ . In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na ϩ , maximal charge translocation (Qmax) was Ϸ25 nC, around a midpoint (V0.5) at Ϫ9 mV, and with apparent valence ϷϪ1. Qmax was conserved upon progressive removal of Na ϩ , whereas V0.5 shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na ϩ partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease. cotransporters; sodium dependent; intestinal absorption; model simulation; renal reabsorption; Xenopus oocyte VITAMIN C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant scavenger of free radicals and as a cofactor in many enzymatic reactions (3,6,10,26,29). It cannot be synthesized in Homo sapiens and must be derived from the diet. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial Na ϩ -dependent Lascorbic acid cotransporter SVCT1 (reviewed in Refs. 32 and 35), the product of the SLC23A1 gene. Selective sorting of SVCT1 protein to the apical membrane has been demonstrated in cultured cell lines (Caco2 and MDCK) of intestinal and renal origin (2, 23).We and others previously demonstrated that human SVCT1 is a Na ϩ -dependent transporter that favors L-ascorbic acid over D-isoascorbic acid and that the oxidized form of vitamin C, dehydroascorbic acid, is excluded (5, 34). Here we explored its molecular mechanisms by testing the hypothesis that the Na ϩ dependence and rheogenicity of SVCT1 arise from...

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Section: Discussionmentioning

confidence: 99%

Section: Heterologous Expression Of Human Svct1 In Xenopus Oocytesmentioning

confidence: 99%

Section: Heterologous Expression Of Human Svct1 In Xenopus Oocytesmentioning

confidence: 99%

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Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Mackenzie¹,

Illing²,

Hediger³

2008

American Journal of Physiology-Cell Physiology

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“…The cloning of SGLT1 and its over-expression in Xenopus laevis oocytes allowed for a detailed characterization of the kinetics of cotransport (Hediger et al, 1987;Parent et al, 1992a,b;Loo et al, 1993;Hazama et al, 1997;Loo et al, 1998). Electrophysiological and tracer uptake experiments in intact oocytes expressing SGLT1 focused on the forward mode, where the substrates are transported from the extracellular to intracellular compartments.…”

Section: Introductionmentioning

confidence: 99%

“…A 6-state kinetic model (see Fig. 6) has been proposed for SGLT1 that accounts for the presteady-state and steady-state kinetics of the transporter operating in the forward mode (Parent et al, 1992b;Hazama et al 1997;Meinild et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Kinetics of the Reverse Mode of the Na+/Glucose Cotransporter

2005

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This study investigates the reverse mode of the Na + /glucose cotransporter (SGLT1). In giant excised inside-out membrane patches from Xenopus laevis oocytes expressing rabbit SGLT1, application of α-methyl-D-glucopyranoside (αMDG) to the cytoplasmic solution induced an outward current from cytosolic to external membrane surface. The outward current was Na + -and sugar-dependent, and was blocked by phlorizin, a specific inhibitor of SGLT1. The currentvoltage relationship saturated at positive membrane voltages (30-50 mV), and approached zero at − 150 mV. The half-maximal concentration for αMDG-evoked outward current ( ) was 35 mM (at 0 mV). In comparison, for forward sugar transport was 0.15 mM (at 0 mV). was similar for forward and reverse transport (≈35 mM at 0 mV). Specificity of SGLT1 for reverse transport was: αMDG (1.0) > D-galactose (0.84) > 3-O-methyl-glucose (0.55) > D-glucose (0.38), whereas for forward transport, specificity was: αMDG ≈ D-glucose ≈ D-galactose > 3-Omethyl-glucose. Thus there is an asymmetry in sugar kinetics and specificity between forward and reverse modes. Computer simulations showed that a 6-state kinetic model for SGLT1 can account for Na + /sugar cotransport and its voltage dependence in both the forward and reverse modes at saturating sodium concentrations. Our data indicate that under physiological conditions, the transporter is poised to accumulate sugar efficiently in the enterocyte.

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Patch Clamp Analysis of Transporters Via Pre‐Steady‐State Kinetic Methods

Grewer

2015

Pumps, Channels, and Transporters

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Presteady-State Currents of the Rabbit Na + /Glucose Cotransporter (SGLT1)

Cited by 96 publications

References 20 publications

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Kinetics of the Reverse Mode of the Na+/Glucose Cotransporter

Patch Clamp Analysis of Transporters Via Pre‐Steady‐State Kinetic Methods

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Presteady-State Currents of the Rabbit Na + /Glucose Cotransporter (SGLT1)

Cited by 96 publications

References 20 publications

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Kinetics of the Reverse Mode of the Na+/Glucose Cotransporter

Patch Clamp Analysis of Transporters Via Pre‐Steady‐State Kinetic Methods

Contact Info

Product

Resources

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Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1